text:
|Method and apparatus for measuring binding affinity
|Fodor et al.
|Fluid handling in mesoscale analytical devices
|Wilding et al.
|Mesoscale sperm handling devices
|Kricka et al.
|Positive flow control in an unvented container
|Peptide library and screening method
|Schatz et al.
|Spatially-addressable immobilization of anti-ligands on surfaces
|Barrett et al.
|Micropipette adaptor with temperature control for PCR amplification
|Gravity assisted collection device
|Containment cuvette for PCR and method of use
|Schnipelsky et al.
|Micromechanical thermoelectric sensor element
|Higashi et al.
|Sensor devices
|Clark et al.
|Device for processing biological specimens for analysis of nucleic acids
|Apparatus for repeated automatic execution of a thermal cycle for treatment of samples
|Capillary flow device
|Hillman et al.
|Biosensors employing electrical, optical and mechanical signals
|Reaction vessel with a rocking base
|Capillary flow device
|Hillman et al.
|Device for use in chemical test procedures
|Shanks et al.
|Capillary flow device
|Hillman et al.
|Multisource device for photometric analysis and associated chromogens
|Eberly et al.
|Diamond film thermistor
|Kobashi et al.
|Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor
|Cozzette et al.
|Method and apparatus for using ultrasonic energy for moving microminiature elements
|White et al.
|Electrical biosensor containing a biological receptor immobilized and stabilized in a protein film
|Taylor et al.
|Sulfate reducing bacteria determination and control
|Gawel et al.
|Enzymatically amplified piezoelectric specific binding assay
|Ward et al.
|Capillary flow device
|Hillman et al.
|Method of manufacturing radiation detector window structure
|Perkins et al.
|Capillary flow device
|Hillman et al.
|Capillary flow device
|Hillman et al.
|Rapid assay processor
|Ebersole et al.
|Slide valve
|Diagnostics testing devices and processes
|de Zabala et al.
|Miniature reaction container and a method and apparatus for introducing micro volumes of liquid to such a container
|Low et al.
|Rapid assay processor.
|Temperature control device and reaction vessel.
|Reaction vessel.
|August, 1990
This is a division of application Ser. No. 08/338,703 filed Nov. 10, 1994, now pending.
placing a first nucleic acid-containing sample and a second nucleic-acid-containing sample into an array comprising a solid substrate, a plurality of wells and channels, wherein at least one channel is formed in the upper surface of the substrate and is situated above another channel formed in the lower surface of the substrate, so as to form crossovers comprising crossing but non-intersecting channels;
moving said first sample into a first well for isolating nucleic acid;
moving said second sample into a second well for isolating nucleic acid;
moving the isolated nucleic acid from the first sample to a third well;
moving the isolated nucleic acid from the second sample to a fourth well;
amplifying the nucleic acid in the third and fourth wells by cycling the temperature of the third and fourth wells;
moving the amplified nucleic acid in the third well to a fifth well for assay;
moving the amplified nucleic acid in the fourth well to a sixth well for assay; and
assaying the amplified nucleic acid.
placing a first nucleic acid-containing sample and a second nucleic acid-containing sample into an array comprising a solid substrate, a plurality of wells and channels, wherein at least one channel is formed in the upper surface of the substrate and is situated above another channel formed in the lower surface of the substrate, so as to form crossovers comprising crossing but non-intersecting channels;
separately isolating nucleic acid from the first and second sample;
separately amplifying the nucleic acid from the first and second sample; and
assaying the amplified nucleic acid.
This invention relates to a system comprising a partitioned microelectronic and fluidic array. More particularly, this invention relates to a system including an array of microelectronic and fluid transfer devices for carrying out various processes, including syntheses, screening and chemical diagnostic assays, in parallel, and method of making the array.
Traditional methods of making a homologous series of compounds, or the testing of new potential drug compounds comprising a series of like compounds, has been a slow process because each member of the series or each potential drug must be made individually and tested individually. For example, a plurality of potential drug compounds is tested by using an agent to test a plurality of materials that differ perhaps only by a single amino acid or nucleotide base, or have a different sequence of amino acids or nucleotides.
Recently the process has been improved somewhat by combining the synthesis of various compounds having potential biological activity, for example, and traditional semiconductor techniques. A semiconductor or dielectric substrate for example is coated with a biologic precursor having amino groups with a light-sensitive protective chemical attached thereto, and a series of masks are placed over the substrate, each mask having an opening. A coupling agent, such as a photosensitive amino acid, is illuminated through the opening, forming a particular compound by reaction with the amino compound. Additional masks are used with different coupling agents to form an array of different peptides on the substrate which array can then be tested for biologic activity. Suitably this is done by exposure of the array to a target molecule, such as an antibody or a virus. The array is exposed to a biologic receptor having a fluorescent tag, and the whole array is incubated with the receptor. If the receptor binds to any compound in the array, the site of the fluorescent tag can be detected optically. This fluorescence data can be transmitted to a computer which can compute which compounds reacted and the degree of reaction. This technique permits the synthesis and testing of thousands of compounds in days rather than in weeks or even months.
However, the synthesis of each coupling reaction is not always complete, and the yield decreases as the length of the biopolymer increases. The process of aligning a plurality of masks and forming openings in the masks in sequence requires careful alignment and takes time.
The above synthesis is made possible by two other recent technical developments that allow various manipulations and reactions on a planar surface. One is the detection and analysis of DNA fragments and their identification by reaction with specific compounds. Probes, RNA and DNA fragments can be resolved, labeled and detected by high sensitivity sensors. The presence or absence of DNA fragments can identify diseased cells for example.
Another step forward is the ability to separate materials in a microchannel, and the ability to move fluids through such microchannels. This is made possible by use of various electro-kinetic processes such as electrophoresis or electro-osmosis. Fluids may be propelled through very small channels by electro-osmotic forces. An electro-osmotic force is built up in the channel via surface charge buildup by means of an external voltage that can "repel" fluid and cause flow. This surface charge and external voltage produces an electro-kinetic current that results in fluid flow along the channel. Such electro-kinetic processes are the basis for a device described by Pace in U.S. Pat. No. 4,908,112 for example.
Thus real progress has been made using electrophoresis and/or electro-osmosis to move very small amounts of materials along microchannels. Such movement can be used for synthesizing very small samples of potential drug compounds in an array and testing very small amounts of materials for bioactivity. Further progress in fully automating the fluidic processes will result in the synthesis and testing of vast numbers of compounds for bioactivity of all types, which information can be made available for future drug selection and will greatly reduce the time and expense of such testing.
The system of the invention comprises a device array of micron sized wells and connecting channels in a substrate that interfaces with a station for dispensing fluids to and collecting fluids from, the array, and for performing electro-optic measurements of material in the wells. The station is also connected to control apparatus means to control the fluid flow to the channels and wells and to collect measurement data from the substrate. The above elements are interdependent and together can perform a variety of tasks in parallel.
The individual wells of the array and their sequence can be varied depending on the synthesis or analysis to be performed. Thus the function of the arrays can be readily changed, with only the additional need to choose suitable interface means for monitoring and controlling the flow of fluids to the particular array being used and the test or synthesis to be performed.
In one embodiment the above system can be used to perform various clinical diagnostics, such as assays for DNA in parallel, using the known protocols of the polymerase chain reaction (PCR), primers and probe technology for DNA assay. In another embodiment the above system can be used for immunoassays for antibodies or antigens in parallel for screening purposes. In still other embodiments, the synthesis of a series of chemical compounds, or a series of peptides or oligonucleotides, can be performed in parallel. Each well in the array is designed so to accomplish a selected task in appropriate modules on a substrate, each module containing the number of wells required to complete each task. The wells are connected to each other, to a sample source and to a source of reagent fluids by means of connecting microchannels. This capability permits broad based clinical assays for disease not possible by sequential assay, permits improvement in statistics of broad based clinical assays such as screening of antibodies because of the parallelism, permits a reduction in costs and an improvement in the speed of testing, and permits improved patient treatments for rapidly advancing disease.
The array is formed in a suitable dielectric substrate and the channels and wells are formed therein using maskless semiconductor patterning techniques. The station and control means, such as a computer, use existing technology that includes commercially available apparatus.
The present device array uses active control to move fluids across the array, reducing the time required for synthesis and screening. Further, large biopolymers of all types can be synthesized and processed while maintaining high purity of the synthesized compounds. The present microlaboratory arrays may be fully automated, enabling the rapid transfer of samples, precursors and other movement of fluids into the array, from one well to another well, and to enable the measurement of assays and the complete control of processing parameters such as temperature control.
FIG. 1A is an exploded schematic diagram of the parts of the system of the invention adapted for performing clinical assays.
FIG. 1B is a top view of a substrate of the invention illustrating a single module formed therein.
FIG. 2 is an exploded top view of an illustrative module of the invention.
FIG. 3 is an exploded cross sectional view of the optical transmission and detection system of the invention.
FIG. 4A is a cross sectional view of a well embodiment of a microlaboratory disc of the invention.
FIG. 4B is a cross sectional view of another well embodiment of a microlaboratory disc of the invention.
FIG. 5A is a cross sectional view of a portion of a module of a microlaboratory disc illustrating devices in typical wells.
FIG. 5B is a cross sectional view of a portion of a module of a microlaboratory disc covered with a cover plate.
FIG. 6A is a cross sectional view of a microlaboratory disc illustrating additional wells having preformed devices therein together with an optical system interface.
FIGS. 6B and 6C illustrate a valve situate in a channel adjacent to a well in the open and closed positions respectively.
FIG. 7A is an exploded schematic view of another embodiment of the present invention adapted to perform immunological assays.
FIG. 7B is a top view illustrating a module on the microlaboratory disc of FIG. 7A.
FIG. 7C is a cross sectional view of a control means for moving fluids in the channels and from one well to another.
FIG. 8 is a schematic view of another embodiment of a microlaboratory array suitable for carrying out the parallel synthesis of proteins and oligonucleotides.
FIG. 9 is a schematic view illustrating a modified station for the system of FIG. 8.
FIG. 10 is a schematic view of a further embodiment of a microlaboratory array suitable for carrying out the synthesis of a large number of small molecules in parallel.
FIGS. 11A, 11B and 11C are cross sectional views illustrating the steps needed to form cross over channels in the substrate.
FIG. 11D is a top view of a cross-over channel in the substrate.
FIG. 12 is a top view of a channel "gate" electrode to control flow in a channel by electro-osmosis.
The invention will be first described by reference to FIG. 1A, which illustrates the parts of an illustrative system of the invention configured to perform DNA screening diagnostics.
The system of FIG. 1A includes a computer 10, electrically connected via line 11 to peripheral apparatus, such as a modem or printer 12, which computer 10 is programmed to give instructions to a microlaboratory disc 14 and to record test results obtained therefrom. The computer 10 is electrically connected to a station 16 via line 15. The station 16 includes a microlaboratory disc support 18, support tubing 20 for loading test materials and reagents onto the microlaboratory disc 14, pumps 22 for moving fluids to particular destinations on the disk 14, one or more light sources 24, an optical fiber 25 and one or more light detectors 26. The optical fiber 25 is operative to transmit light from the light source 24 to the detector 26. One or more containers 28 for waste fluids and the like are also housed in the station 16.
A small volume of a fluid to be tested, such as a whole blood or other DNA-containing fluid sample, is loaded into the system via a loading system 30. The system 30 may house one or more capillary tubes 32, illustrated in FIG. 3, containing a sample which is connected to a loading capillary channel 34 etched into the surface of the microlaboratory disc 14. The loading capillary tube 32 is inserted into the capillary loading channel 34 horizontally. The sample is moved into the capillary loading channel 34 and the fluid sample is then moved to a first well 36 on the disc 14. Alternately, a loading channel 50 for vertical insertion into the loading channel 34 can also be used to load the sample. FIG. 1B and FIG. 2 illustrate a particular module comprising a plurality of wells 36, 40, 42 and 44 connected by a channel 38, shown by a dashed line, on a microlaboratory disc 14 of the invention. Each module is connected to a well 46 that collects excess or waste fluids from the respective module. These waste fluids are collected and moved to the waste containers 28 in the station 16.
The sample is treated sequentially in a series of wells including first well 36. For example, in the first well 36 the whole blood sample is transferred from the capillary loading channel 34, filtered and lysed to separate the white and red corpuscles, and the DNA is isolated from the white blood cells. The DNA sample is then moved out of the first well 36 through a connecting channel 38 that connects all of the wells of a single module, and into a second well 40. In the second well 40 the DNA is separated into single strands and amplified using the well known PCR method. The treated sample is then moved out of the second well 40 via the connecting channel 38 and into a third well 42. In the third well 42 the DNA is assayed by known probe hybridization techniques. The DNA assay is detected and evaluated in the fourth well 44. Thus the determination of DNA in a particular blood sample is performed in a series of four wells connected by a channel. Lastly excess reagents and the like are collected in the fifth well 46 that is common to all of the modules in the microlaboratory array 14, and is transferred into the waste collection system 28 of the station 16.
The combination of a loading channel 34 or 50, the wells 36, 40, 42, 44 and 46 and the connecting channel 38 make up one module 48 on the test microlaboratory disc 14. A single module on a microlaboratory disc 14 is shown in top view in FIG. 1B within the dashed lines. As will be explained hereinbelow, a plurality of modules are formed in the microlaboratory disc 14 so that tests can be performed on a large number of the modules 48 in parallel.
The particular sequence of channels and wells of the module shown in FIGS. 1A, 1B and FIG. 2 lends itself to the detection of pathogenic bacteria in blood or other DNA-containing fluids using the DNA assay protocols of Greisen et al, see "PCR Primers and Probes for the 16S rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria Found in Cerebrospinal Fluid", J. Clinical Microbiology 32 (2), pp 335-351 (1994). Since the disc 14 can be arranged to form a plurality of up to 1500 parallel modules, broad DNA screening can be conducted in parallel that might not be possible using a system of sequential assays. Since a large array of parallel tests can be done at the same time, the time required to obtain meaningful results is also reduced, and consequently costs are reduced. Further, the statistics of broad based screening can be improved. In addition the system of the invention provides the ability to detect mutant DNA such as is found in oncological diseases.
The station 16 includes a fiber optic assembly 25 and one or more light sources 24 and one or more detectors 26 (not shown) that address the system loading channel 34 or 50 and measures the transmittance or absorbance of material in the channel 34 or 50 and the first well 36, such as a blood or other fluid sample. The fiber optic assembly 25 can verify the presence or absence of materials in the channel 34 or 50 or the well 36, and quantify their amounts by transmitting the measurement data to the computer 10. Suitable lasers and photodetectors are available commercially. Fiber optic adaptors to support the optical fiber are commercially available. These adaptors may also include a lens for efficient transfer of light from the light source into the fiber.
Preselected substrates, designed either for modular parallel or array processing, can be placed on the substrate holder 18 in the station 16. After suitable modification of the software in the computer 10 or choice of other driving means able to monitor a particular sequence of steps for control of reagents to carry out the processes in each module of the microlaboratory 14, entirely different tests and processes can be carried out in the system of the invention. Thus the microlaboratory array 14 is individually designed for a variety of assay, measurement and synthesis tasks, and only the module configuration and the driving means needs to be changed in the system when a different task is to be performed.
A circuit of thin film transistors (not shown) can be formed on the front or back of the glass or other substrate 14 to provide power to the wells via leads and electrodes explained further hereinafter, and to connect them with the driving means such as the computer 10, so as to move liquids along the array. Pins can also be formed in the glass substrate which are addressable by logic circuits on the support 18 that are connected to the computer 10 for example. These transistors and logic circuits will also be in contact with the substrate 14 on the support 18 to provide an interface between the microlaboratory disc 14 and the computer 10.
The system loader 30 is situated outside of the station 16 and may be connected to a loading capillary tube 32, as shown in FIG. 3, suitably having an inner diameter of about 200 microns and an outer diameter of about 600-700 microns. The capillary tube 32 can be pretreated to eliminate surface adsorption of proteins and related bio materials in a known manner, for example similar to the methods disclosed by Cobb, "Electrophoretic Separations of Proteins in Capillaries with Hydrolytically Stable Surface Structures", Anal. Chem. 62, (1990) pp 2478-2483. All of the sample material is loaded into the system via the system loader 30. The system capillary tube 32 can be loaded horizontally directly into the microlaboratory disk 14 via the loading channel 34. The sample is identified, as with a bar code or other high density code affixed to the loading capillary tube 32 as by its position on the disc array. As the sample loading tube 32 is inserted into the loading channel 34, a sealant, which can be adhered to the edge of the capillary sample tube 32 or to the loading channel 34, seals the capillary tube 32 to the channel 34. Alternately, the sample capillary tube 32 can be inserted into a separate loading channel 50 vertically, which permits a smaller loading channel to be used, but the sample may enter into the connecting channel 38 through force of gravity rather than via a controlled pump feed from the pumps 22 in the station 16. This alternate configuration 50 is also shown in FIGS. 1-3.
The heart of the present system is the parallel modular microlaboratory disc 14, shown in a side view in FIG. 1A and in a top view in FIG. 1B. FIGS. 1B and 2 illustrate a single module on a microlaboratory disc 14, which is suitably about 8.5 cm in diameter.
Each module of the microlaboratory array is made up of one or more wells connected by a channel, in turn connected to the loading capillary tube. The microlaboratory disc 14 itself can be made from glass, fused silica, quartz or a silicon wafer for example, suitably about 1 millimeter thick, that is able to be finely and controllably etched using semiconductor techniques and a suitable etchant such as HF. High quality glasses such as a high melting borosilicate glass or a fused silica, will be preferred for their UV transmission properties when any of the processes or measurements carried out in the wells or channels use light based technologies. The module 48 illustrated in FIG. 1B comprises four connecting wells, but this is by way of example only, and more or fewer wells can be present depending on the tests or syntheses to be performed in each module, and the number of steps required to perform them, as will be further described hereinbelow.
The desired number of wells are etched into the microlaboratory disk sufficient to perform the sequence of steps to be used for testing or synthesis for that particular microlaboratory disc. All of the wells in each module are connected together via one or more channels. The modular design permits efficient placement of each module on the disc substrate. When only a few wells are required for each module, two modules can be formed in each radial slice of the disc (not shown), thereby doubling the number of modules that can be etched into a single microlaboratory disk 14. Thus for an 8.5 cm diameter disk, an array of 267 single modules or 534 double modules can be readily made. When the sample is inserted vertically into the loading channel 50, which takes up less area of the substrate, additional modules can be accommodated on a single microlaboratory disc, and up to 1500 modules can be formed on a single microlaboratory disk 14. Each of the modules is connected to the system loading system 30, thus permitting parallel processing in all of the modules.
Excess fluids and waste materials for all of the modules are passed into a center well 46. A plurality of wells 46 may be connected to each other by means of a common channel 47 (FIG. 1B). A sealed tubing on the back of the substrate 14 provides an exit channel for these excess fluids that can be passed into the waste container 28 in the station 16. Thus only one exit channel 47 needs to be provided for each microlaboratory array.
The wells of the microlaboratory disc 14 can be made by the following procedure. A glass substrate disc 14 is coated sequentially on both sides with a thin chromium layer and a gold film about 1000 angstroms thick in known manner, as by evaporation or chemical vapor deposition (CVD), to protect the disc from subsequent etchants. A two micron layer of a photoresist, such as Dynakem EPA of Hoechst-Celanese Corp. is spun on and the photoresist is exposed, either using a mask or using square or rectangular images, suitably using an MRS 4500 panel stepper of MRS Technology, Inc. After developing the resist to form openings therein, and baking the resist to remove the solvent, the gold layer in the openings is etched away using a standard etch of 4 grams of KI and 1 gram of iodine (I2) in 25 ml of water. The underlying chromium layer is then separately etched using an acid chromium etch, such as KTI Chrome Etch of KTI Chemicals, Inc. The glass substrate is then etched in an ultrasonic bath of HF--HNO3 --H2 O in a ratio by volume of 14:20:66. The use of this etchant in an ultrasonic bath produces vertical sidewalls for the various wells. Etching of the wells is continued until the desired depth of the well is obtained, suitably about 200-750 microns deep, depending upon the functions to be performed in the well.
The connecting channel 38, suitably about 200 microns in width and 50-100 microns deep, is then etched between the wells, when the remaining photoresist and chromium-gold layer is removed.
Fluid material may be transmitted to the various wells from the first well 36 or from the loading channel 34 by various methods. An external mechanical pumping system 22 that can deliver fluid in reproducible and accurately controlled amounts and that can maintain the purity and sterility of test liquids can be employed. The 205U multichannel cassette pump available from Watson-Marlow, Inc is a suitable pump. Alternatively, miniaturized mechanical pumps, based on microelectromechanical systems (MEMS) can be used. These miniature pumps may be internal or external to each well 36, 40, 42 and 44. Such pumps have been reported by Shoji et al, "Fabrication of a Micropump for Integrated Chemical Analyzing Systems", Electronics and Communications in Japan, Part 2, 70, pp 52-59 (1989). Other suitable pumping means to move fluids through microchannels include electro-kinetic pumps as reported by Dasgupta et al, see "Electroosmosis: A Reliable Fluid Propulsion System for Flow Injection Analysis", Anal. Chem. 66, pp 1792-1798 (1994), or electrophoresis methods, which require inert metal electrodes, can also be used. In the latter case, a gold electrode can be deposited in the various wells of a module, as shown for example as metal layer 54 in the bottom of the first well 36, as shown in FIG. 4A. The electrode 54 is connected via leads 55 to a circuit or other electrical connection 56.
When large amounts of fluid need to be passed through the connecting channel 38, such as diluents, buffer solutions, rinse solutions and the like, an external mechanical pump 22 in the station 16 will be used to pass these solutions through the channel 38 into the desired well. When smaller amounts of material are required, an on-disk internal pumping system can be used for greater efficiency. Further, prepackaged chemicals sealed in plastic release containers can be deposited in the various wells for particular tests, as required.
In the case where the temperature of a particular well is to be monitored or changed, a means of heating or cooling the well is built into the well, as will be further explained below with reference to FIG. 4B.
The first well 36 in this example is the sample preparation well. A thin film of a suitable metal oxide 57, such as tin oxide or indium tin oxide, is deposited onto the well material and is connected by means of an electrically conductive metal connection 58 to the end or outer edge of the well 36. The tin oxide coating 57 serves as a heater element for the well 36. The well 36 also has a surface bimetal film 59 and leads 60, suitably made of chromel-alumel alloys, forming a thermocouple to measure the temperature in the well when a source of current is applied to the tin oxide coating 57 and to the leads 58. A voltage applied to the well 36 via electrodes 56 deposited on the backside as shown, or preferably on the frontside of the microlaboratory disc 14, regulates the temperature in the well. The amount of current applied can be regulated by the computer 10 in response to the temperature measured through the leads 60.
The first well 36 of the present module 48 also contains a blood affinity binding material, (not shown) such as Leukosorb™ available from Pall Co. and commercially available solutions to lyse the red and white blood cells of the sample and to carry the DNA from the first well 36.
Thus the devices and certain reagents needed for each well will be preformed and loaded into the well or directly pumped into the well from a common source. Fluids can be preloaded into the well either from a reservoir in the station 16 leading to the loading capillary tube 32 connected to the microlaboratory disc 14, or a reservoir and channel into the well may be formed adjacent to the well in which the fluids will be used.
Additional devices can be built into the wells. For example, a means of stirring reactants can also be built into a well using alternating fields. For example, two or more electrodes can be evaporated into each side of a well and connected to external leads, in turn connected to a source of alternating current. The alternating fields will provide reversible magnetic fields to move paramagnetic particles in the wells and to cause, as well, fluid movement in the well.
As shown in FIGS. 5A, paramagnetic beads 61 are deposited in the second well 40 of the present module 48, to bind DNA material and to move the DNA to the succeeding wells for amplification, detection and assay. These beads are commercially available for PCR protocols from Bang Laboratories of Carmel, Ind. for example. The well 40 is also connected by means of leads 66 to the electrodes 56.
When all of the wells have been prepared and loaded, a glass cover plate 63 is affixed to the microlaboratory disc 14, as shown in FIG. 5B, to complete a capillary structure for the connecting channel 38 and to ensure that fluids in the wells do not evaporate. The cover plate 63 can be made of the same or different material as the microlaboratory disc, but the thermal coefficient of expansion of the cover plate 63 and the material used to make the microlaboratory disc 14 must be similar. The sealing temperature required to seal the cover plate 63 to the disc 14 must also be below the flow temperature of the disk material to prevent distortion of the etched channels and wells. The use of electric fields to seal the cover plate to the disk is suitable because sealing will take place at a lower temperature than the flow temperature, e.g., about 700° C. or less, which is well below the flow temperature of silicon, about 1400° C., or of Corning 7059 glass (about 844° C.), available from Corning Glass Co., for example.
The invention will be further explained in terms of examples that describe different modules for performing different processes, such as assays and syntheses, simultaneously and in parallel. However, the invention is not meant to be limited to the details described therein and additional processes and syntheses can be readily performed by appropriate changes to the number of wells in a module, the devices formed in or adjacent to the wells, the number and type of devices needed to pass fluids into and out of the wells, the number and type of processing-type devices built onto the disc other than the modules, and the like.
The sample fluid is passed into the loading channel 34 or the loading channel 50 as explained above. The sample then moves by application of an electric field or is moved by a pump 22 into the first well 36 through the channel 38, where the separation of the sample, e.g., filtration, lysation and DNA separation, takes place. The first well 36 is fitted with a means of heating and temperature control, as shown in more detail in FIG. 4. A layer of tin oxide 57 is first deposited in the well 36 by CVD. A bilayer film 59 is deposited over the tin oxide film 57 in the well 36, and a metal connection 60 is deposited along a sidewall of the well. Electrodes 56 and 60 are formed on the backside of the microlaboratory disc 14, and leads 58 and 60 connect the thermocouple 59 to the external contacts 56. The current in the leads 58 and the voltage from the leads 60 are monitored and controlled by the computer 10.
The well 36 is also preloaded or post loaded with a blood affinity binding material such as Leukosorb™ media from Pall BioSupport Div. Other commercially available materials can also be used as desired for the same purpose. The amount of Leukosorb B employed depends on the area in the first well 36. The Leukosorb B filters the blood cells from the blood serum sample. A known buffer solution is then passed into the first well 36 by means of the channel 38 in an amount sufficient to assist in lysing and washing the red corpuscles in the sample. A white corpuscle lysis buffer solution, for example a solution of 50 millimoles of KCl, 10 millimoles of Tris HCl having a pH of 8.3, 2.5 millimoles of MgCl2, 0.45% Nonidet P40 and 60 ml of Proteinase K, is then passed into the first well 36 via the channel 38 in an amount sufficient to lyse the white corpuscles. The first well 36 is then heated to 55° C. for fifteen minutes to cause the desired reaction. The cell was then heated to 100° C. for about 10 minutes to inactivate the proteinase K following known procedures.
Because of the high temperatures required in the last two steps, a significant vapor pressure may develop in the first well 36, causing a back pressure in both directions--back toward the sample loading channel 34 and forward to the succeeding second well 40. Thus preformed valves 62 and 63 as shown in FIG. 6A, formed of bimetallic materials as described by Jerman et al, "Understanding Microvalve Technology", Sensors, September 1994 pp 26-36 are preloaded into the channel 38. These materials have a thermal expansion mismatch. When the temperature in the well 36 is low, the ball valve 62 is in its normal position permitting free flow of fluids into the well 36, see the arrow shown in FIG. 6B. As the temperature in the well 36 increases, the ball valve 62 moves to a cooler position, as shown in FIG. 6C, blocking the channel 38 and isolating the well 36 from the channel 38, thereby preventing fluids from passing into and out of the first well 36. By proper placement of the well heater 57, the temperature gradient from the heated well 36 will be sufficient to open and close the valves 62, 63, thus eliminating the need for separate electrical, thermal or mechanical control of the valves 62, 63.
However, other means may be employed as desired for closer control of the temperature and pressure in the well 36. A simple design comprises a small micron size sphere 64, such as one made of quartz or polytetrafluoroethylene polymer for example, which may be confined in the channel 38 to act as a conventional check valve, i.e., to prevent backward flow into the channel 38. However, there is no check on the flow forward into the succeeding well 42 with this design, which may be disadvantageous for certain applications and combinations of sequential wells. This check valve 64 can be implemented as an insulating or magnetic sphere, and by application of externally applied electrostatic or magnetic fields, cause the valve to open or close. Such a design will permit closure of the ball valve 64 on demand, permitting control of liquids both forward and backward in the channel 38. Such a design may be particularly advantageous when a synthesis procedure is carried out on the microlaboratory disc 14.
Still referring to FIG. 6A, the prepared and treated blood sample is then transferred to the second well 40 for PCR amplification of the obtained DNA sample from the first well 36. This well 40 contains a layer of paramagnetic beads 61 to bind with the DNA material moved into the well 40. A minimum of about 10 nanograms of DNA are required for this assay. The amount actually in the well 40 can be determined by the electrooptic light source 24 and detector 26 located in the station 16. An optical fiber 25 of the light source 24 is connected to the well 40, and can be an optical fiber 25 of 200, 400 or 600 micron diameter to provide 260 nm radiation for the detection of DNA in this example. Optical fibers transmitting at 250-260 nm are commercially available. The optical fiber 25 of FIG. 6A which is connected to the detector 26 collects and transmits the light measured in the well 40 to an ultraviolet sensitive silicon photodetector 26 having good stability. Suitable detectors include thermoelectrically cooled UV enhanced silicon photodetectors, or miniaturized UV sensitive photomultipliers which are also commercially available. The photomultiplier-type detector has greater sensitivity. The absorbance of DNA is known and is set forth in the Table below.
|Absorbance Species at 260 nm Concentration mg/ml
Double stranded DNA
1 50
Single stranded DNA
1 33
Single stranded RNA
1 40
A well known means of assaying DNA is the hybridization technique. The third well 42 is used for this purpose. The well 42 will be fitted by hybridization probes which are unique to the source of DNA and its diagnostic scope. The desired probes may be directly synthesized in the well 42 or commercial sources can be used.
The method most suitable for the microlaboratory array disc 14 for assaying DNA is the use of a non-radioactive tag, which can contain a fluorescent dye, for example for the Phycobiliprotein, or other class of protein. These materials absorb light above 600 nm. A compact solid state laser 24 emitting in the 600-650 nm range situate in the station 16 can be used to produce fluorescence in the tag. The probe in this example will emit above the exciting wavelength of 600 nm, i.e., in the 600-800 nm range. The Phycobiliprotein fluorescent tag agents are commercially available, e.g., Cy5 ™, from Jackson Immuno-Research Labs. Inc of West Grove, Pa. The fluorescence will be detected by means of the detector 26 also in the station 16. A filter 68 will also be inserted between the photomultiplier photodetector 26 and the laser 24 as shown in FIG. 6 to block the laser radiation.
A suitable detector for the fluorescence emitted by the fluorescent tag is one having high sensitivity and optimum long term stability. Other commercially available means for detection of antibodies other than that described above can also be employed. For example, enzymes that form highly colored products, resulting in detection having excellent sensitivity, or chemiluminescent compounds can be employed and detected optically. Radioisotopes can also be used, but their use is being discouraged to prevent adverse effects on the operator of the tests, and they present disposal problems. The light transmission detection system itself is also located in the station 16.
A second example illustrating the design and utility of a microlaboratory disc 114 is shown in FIGS. 7A and 7B and is one suitable for carrying out a plurality of immunological assays, also in parallel. The station 16, sample loading channel 134, and plurality of wells 136, 140, 142 and 144 connected by a channel 138 are similar to those of Example 1, but the materials and processes carried out in the wells, and the number of wells required, are different.
To carry out an immunological assay, a blood sample or other fluid sample of about 100 nanoliters is filtered to obtain a material having sufficient antibodies so that an assay can be determined using standard methods for detection of select antibodies. The microlaboratory disc 114 is able to process 200-1000 samples in parallel.
The initial transfer of a sample to the microlaboratory array 114 is the same as for Example 1. The first well 136 is loaded with preselected bound antibodies on the cell wall, and, by transfer of appropriate reagents and fluid flow control into each well 136 of the module 148, assays can be performed with that antibody. The flow of reagents and solvent will be controlled by the station 16, and the optical detection system 24, 25 and 26 will be set up for transmittance (absorption) measurements.
A series of different antibodies can be placed in additional wells 140, 142, 144 and 146 for sequential testing for various antibodies in successive wells, as shown in FIG. 7B. The number of wells required will be determined by the number of appropriate reagents and the number of antibodies to be tested.
A control means 171 to control the fluid flow of reagents and solvents will be enabled by element 162, which is a switch or valve that controls the flow of fluid into the channel 138 and into the first well 136. For example, a magnetic sphere can form a ball valve 162 by activation of an electric field by means of the computer 10.
As shown in FIG. 7C, a ball valve 162 is fitted into the channel 138 on both sides of the well 136. A source of current 170 is placed adjacent the channel 138 to control the flow into or out of the well 136, and the backside of the microlaboratory disc 114 will have a lead 172 that connects to the driving method employed, such as the computer 10.
This example, shown in FIG. 8, illustrates a microlaboratory disc 214 that has a modular design that permits the synthesis of a large array of proteins or oligonucleotides in parallel. Such synthesis is a great time saver for drug testing for example, when a whole series of related compounds can be synthesized in each well of an array, and then tested for biologic activity.
For example, the synthesis of solid phase oligonucleotides in a well has been described by Hayakawa et al, "The Allylic Protection Method in Solid-Phase Oligonucleotide Synthesis. An Efficient Preparation of Solid-Anchored DNA Oligomers", J. Am. Chem. Soc., Vol 112, No. 5, 1990 pp 1691-1696, in a sequence of eight steps:
Step 1: washing the well with acetonitrile (CH3 CN)
Step 2: detritylation with 3% Cl3 CCOOH--CH2 Cl2
Step 3: washing the well with acetonitrile
Step 4: adding a coupling agent such as 0.1M phosphoramidite--CH3 CN and 0.5M (4-nitrophenyl)tetrazole--CH3 CN--THF (tetrahydrofuran)
Step 5: washing the well with acetonitrile
Step 6: capping with Ac2 O--2,6--lutidine--THF (1:1:8) and 6.5% 4-(dimethylamino)pyridine (DMAP)--THF
Step 7: oxidizing with 1.1M t--C4 H9 OOH--CH2 Cl2
Step 8: washing the well with acetonitrile to remove excess acid.
After the above 8 steps have been carried out in a reaction well 237 using different protected monomers sequentially to build up lengths of oligomers as desired, additional reagents or solvents can be used to remove protective groups used for synthesis prior to disassociating the synthesized oligomers from their support. The various oligomers in each well can then be prepared for screening. A screening cell 274 can also be built into the well 236 as shown in FIG. 8.
In this design, as shown in FIG. 8, a plurality of modules or wells 236 are connected by means of various channels, and a complete synthesis of different compounds will be carried out in a single well 236. The additional features of this microlaboratory 214 design are in the array design which permits controllable and sequential delivery of various reagents and solvents to each of the wells in parallel. The reagents and solvents can be stored in reservoirs 276, 278, 280 and 282 built into the microlaboratory array, or they may be delivered from reservoirs external to the microlaboratory array, for example in the station 16, as desired.
FIG. 8 illustrates a suitable design for a microlaboratory array wherein reservoirs 276, 278, 280, 282 for reagents and solvents are included in the substrate; and FIG. 9 is a partial schematic view of a system for synthesis wherein reservoirs 276, 279, 280 and 282 are added to the station 16, each connected to a fluid pump 22 for delivery to the wells of each module.
Each well in this array has a plurality of horizontal channels 238, 239, 241, 243, 245 entering and exiting each well 236, and a plurality of vertical channels 248, 250, 252 and 254, each of which leads to a different reservoir. The reagents and solvents move along the channels by means of gating electrodes, as shown in FIG. 7C, that move the fluid along the channels.
As shown in FIG. 8, reagents are delivered in parallel from the different reservoirs 276, 278, 280 and 282 into channels 238, 239, 241, 243 respectively. Each of these channels has a built in gating electrode 162 to monitor the flow of fluids from the different reservoirs into the wells 236 as required. In this embodiment, since all of the synthesis reactions take place in a single well 236, each well 236 is placed in an array and one or more channels lead into and out of each well 236 for delivery of reagents in parallel or serially. Thus instead of a modular design, when sequential wells are used for the various steps of the process being carried out, an array of like wells 236 is used but a different chemical synthesis is carried out in each well. The overall design is no longer a plurality of modules, but an array of wells, and hundreds of compounds can be synthesized on a single microlaboratory substrate 214.
After all the synthetic steps are completed, the syntheses can be monitored or screening can be conducted by passing a sample into a screening cell 274 which can be built into the well 236.
This type of array can be used to synthesize a large number of oligomers and peptides which follow a repetitive sequence of steps as above.
This microlaboratory array of this example 314 is also designed to produce an array of small molecules, such as a plurality of different alkyl compounds. Since such an array depends on the delivery of a different starting material for each compound, e.g., methyl-substituted, ethyl-substituted, n-propyl-substituted, iso-propyl substituted compounds and the like, a large array of starting material reservoirs or supply sources will be needed so that a different starting material will be delivered to each well. The reactants and solvents and reaction conditions, e.g., heating, will be delivered as required, see Examples 1 and 2.
For example, the synthesis of an array of 1,4-benzodiazeprine compounds can be carried out as follows:
Step a): bind the starting material 4-[4-benzoyl-6-chloro-2-fluorenylmethoxy carbonylaminobenzoyloxy-methyl]ph enoxyacetic acid onto aminomethyl resin or its equivalent as a coating material on the channel surfaces;
Step b): remove the 9-fluorenyl methoxy carbonyl (FMOC) protective group from the bound material using piperidine in DMF, supplied from station reservoir 376 into the wells 338;
Step c): couple an amino acid, alpha-N-FMOC-amino acid (fluoride) from a second reservoir 378 in the well 338;
Step d): remove the FMOC protective group by moving piperidine in DMF through the wells 338;
Step e): forming a cyclic benzodiazepine derivative with 5% acetic acid in DMF from a third reservoir 380 in wells 340;
Step f): alkylate with a lithium salt in DMF from a station reservoir 382 in wells 342 that are fitted with a control for pumps and switches, monitored by the computer 10, to deliver different alkylating agents to the microlaboratory array 314 selectively, as shown in FIG. 10. Thus the "reservoir" for the alkylating agent is in reality a source of a whole series of alkylating agents that can be delivered selectively to a particular well in the array. The fluid delivery to the wells can be controlled by a control means 171 as shown in FIG. 7C. In this manner, a different compound is made in each well. However, since each compound requires several steps for synthesis of the various compounds, several wells may be employed to synthesize each of the compounds and this microlaboratory disc 314 may comprise a plurality of modules as well.
Another feature of the present invention is the fabrication of cross-over channels that permit high density arrangements of wells/modules in an array that can be serviced from more than one channel either to transmit fluids into the well, out of the well, or to transmit the material being treated to a succeeding well. Such cross over channels can be formed in the following manner. Referring to FIG. 11A, a substrate 314 is coated on both sides with a first chromium-gold layer 310A and 310B and a photoresist layer 312A and 312B as described above. The photoresist layer 312A is exposed and developed to form openings 313 for a first channel and the chromium layer 312A is etched away in the opening 313. The glass substrate 314 is then etched through to the chromium-gold layer 310B forming openings 315. The photoresist layers 312A and 312B are re-exposed and developed to form a second pair of openings 316 and 317 on each side of the glass substrate 314. However, the second opening 316 in the layer 312A is perpendicular to the opening 317 in the layer 312B, which is parallel to the openings 315 made originally. The opening 317 is formed adjacent to the openings 315 already formed in the glass substrate 314, as shown in FIG. 11B. The glass substrate is next etched to open a passage between the openings 315 and 317, thereby forming a continuous channel that extends through the glass substrate 314 and across beneath the second perpendicular channel 316 as shown in FIG. 11C. A cover plate 318 will be bonded to both sides of the substrate 314 to enclose the cross-over channels 315-317-315 and 316 on both sides of the substrate 314 and to complete the substrate having crossing but non-intersecting channels, as shown in FIG. 11D.
In order to move materials from the reservoirs into each well of the array, the material from the reservoir can be injected into one or more the channels 316. The material in each channel moves along the channel 316 via capillary action. However, since one or more of the reactions may require more or less time depending on the amount of material employed, or to compensate for faster or slower movement of certain materials through the channels, the material flow in the channels preferably can be controlled by means of a drain electrode deposited in each well, and in each reservoir and in the channel leading from the reservoir to the respective well. By creating a negative charge at the well, the flow along the channel will be stopped, thereby permitting control of reagent feed into each well. This is shown in FIG. 12 which is a top view which shows a drain electrode 440 deposited in the well 436, in a channel 438, and in the reservoir 442.
A desired microlaboratory array can be designed to carry out a wide variety of tests and syntheses rapidly, reliably, inexpensively and in parallel, greatly reducing costs. Further, because of the integration of the microlaboratory array and means of regulating, measuring and recording information, records of tests and screening data are reliably generated and stored. Since genetic screening may become the blood tests of the future, the ability to perform DNA testing rapidly, reliably and at low cost, will be highly advantageous.
Thus although the present invention has been described in terms of particular examples, the invention is not meant to be limited to the details given above. One skilled in the art can readily substitute different mechanical or electrical means of transferring fluids through the channels and wells, of substituting various bonding agents, starting materials and reagents to perform different assays and syntheses, and to form the wells, channels and devices in the channels and wells by alternate methods. For example, integrated circuits can also be employed to move fluids and monitor and measure wells of the arrays using conventional semiconductor techniques and materials. Semiconductor technology can also be used to form leads, electrodes and the like. Thus the invention is only to be limited by the scope of the following claims.


text: Almost a year ago now I started reading the A Song of Ice and Fire series. Currently I'm around 200 pages from completing the third book, A Storm of Swords. While I know I still have another massive tome to go before I'm caught up I wanted to write some commentary after the events of last night's reading. For anyone who hasn't read them yet I'll be sure to clearly label where the spoiler section begins. Don't expect much out of this. Just a few random thoughts.

subset: commoncrawl


text:
Movement Disorders
St. Luke's Movement Disorders Center
Aaron C. Lasker, MD is the Medical Director of the St. Luke's Movement Disorders Center. The team includes fellowship-trained neurologists Nancy Diaz-Pichar, MD and James Shou, MD. This group treats Parkinson's disease, Huntington's disease, restless leg syndrome, essential tremor and other movement and gait disorders. The team also includes functional neurosurgeon Roy Hwang, MD who performs Deep Brain Stimulation (DBS) to treat movement disorders.
Parkinson's Disease
PD STEPS Program
The Support To Empower
Register by calling InfoLink
Parkinson's Disease is a brain disorder that occurs when certain nerve cells in the part of the brain that controls muscles and movement are damaged or die. Normally, these cells produce a chemical (dopamine) that lets the body's muscles move smoothly. According to the National Parkinson Foundation (NPF), the symptoms of Parkinson's disease appear when about 80 percent of these cells are damaged.
Although Parkinson's disease usually appears after the age of 65, 15 percent of people with the disease are younger than 50. It affects men and women at almost the same rate. While there is no test that can diagnose the disease, tests are done to rule out other conditions with similar symptoms.
Medications can be used to ease or control the symptoms of Parkinson's disease. Since most of the symptoms of the disease are caused by a lack of dopamine in the body, the most commonly used medications replace or imitate dopamine. Doctors may suggest:
There are no known ways to prevent Parkinson's disease.
Occurring most often in the hands, tremor is an unintentional, rhythmic, muscle movement involving to-and-fro movements. The tremor also may affect the chin, lips, legs, and trunk. Excessive alcohol consumption or alcohol withdrawal can kill certain nerve cells, resulting in tremor, especially in the hand. Other causes include an overactive thyroid and the use of certain drugs.
Although tremor is not life-threatening, it can cause embarassment and make it harder to perform daily tasks.
There is no cure for most tremors. The appropriate treatment depends on accurate diagnosis of the cause.
Although the most common form occurs in otherwise healthy people, some forms of tremor run in families, while others have no known cause. Tremor may occur at any age but is most common in middle-aged and older persons.
Huntington's Disease
Huntington's Disease is distinguished by uncontrolled movements, loss of intellectual faculties and emotional disturbance. It is caused by genetically programmed degeneration of brain cells (neurons) in certain areas of the brain. A child has a 50 percent chance of inheriting the mutated gene. If a child does not inherit the HD gene, he or she will not develop the disease and cannot pass it to subsequent generations. A person who inherits the HD gene will sooner or later develop the disease.
Early symptoms include mood swings, depression, irritability or trouble driving, learning new things, remembering a fact, or making a decision.
Later symptoms include increasing difficulty with intellectual tasks, feeding and swallowing.
A genetic test along with a complete medical history, neurological tests and laboratory tests will help physicians diagnose Huntington's Disease.
A number of medications are available to help control emotional and movement problems associated with Huntington's Disease. Most of these medications have side effects including fatigue, restlessness or hyperexcitability.
It is recommended that people with Huntington's Disease maintain physical fitness. There is evidence that individuals who exercise and keep active tend to do better than those who do not.
At this time, there is no way to stop or reverse the course of Huntington's Disease. Now that the gene has been located, investigators are continuing to conduct research to better understand how it causes disease in the human body.
Restless Leg Syndrome
Restless legs syndrome is a common neurological disorder distinguished by unpleasant sensations in the legs and an uncontrollable urge to move them for relief. These sensations can range from uncomfortable to irritating to painful.
Lifestyle changes can help those experiencing mild to moderate symptoms.
There is no cure for Restless Leg Syndrome. Symptoms gradually may get worse with age. Current therapies can control the disorder, minimizing symptoms and increasing periods of restful sleep.
Some individuals have experience remissions in symptoms, which may decrease or disappear for short or long periods. However, symptoms eventually reappear.
Dystonias are characterized by sustained muscle contractions that cause twisting and repetitive movements or abnormal postures. These involuntary and sometimes painful movements, may affect a single muscle, a group of muscles or the entire body.
Dystonia can affect many different parts of the body.
There is no single treatment for dystonias. Physicians recommend a number of therapies to reduce or eliminate muscle spasms and pain including:
There is no current evidence to suggest that dystonias can be prevented.


text:
Enhancing the sagebrush sea
Special to the Daily
In many parts of the valley, where soils are dry and well drained, there are patches of a "sagebrush sea." Even though a sagebrush landscape may look bland and monotonous to the casual observer, these fragile and disappearing ecosystems are sometimes teeming with life.
The "sagebrush sea" is a complex living patchwork and home to many native species of wildflowers, mammals, reptiles, amphibians, birds, and insects.
Historically, the "sagebrush sea" covered 270 million acres across the American West and today less than 150 million acres remain. Some sagebrush species have lost 50 percent of their historic native habitat. The threats to sagebrush habitat are many, including the spread of invasive non-native weeds, fire suppression, and a variety of human land uses that cumulatively fragment the "sea" into small bits and pieces, making it difficult for wildlife populations to access. A recent scientific survey of sagebrush around Eagle County discovered that much of the sagebrush is old and declining due to wildfire suppression and other historic land use practices.
Because of the extent of the "sagebrush sea" across the West, the complexity of public and private land ownership patterns, and the dependency of the endangered sage grouse and other obligate wildlife species, sagebrush habitats have become a focal point for large-scale landscape planning, restoration and monitoring. As part of this overall effort, this summer the National Forest Foundation and Vail Resorts are partnering with the U.S. Forest Service and many nonprofit partners and other agencies to restore and enhance the resiliency of local sagebrush habitats. Vail Resorts and National Forest Foundation Ski Conservation Funds are contributing $236,000 to five nonprofit partners conducting work on Forest Service lands, and in the process this funding is also creating summer jobs for 20 youth through the Rocky Mountain Youth Corps and a new internship program at Walking Mountains Science Center.
This is no small effort. Lara Duran, the wildlife biologist at the Eagle-Holy Cross Ranger District based out of Minturn and Eagle, is the point person coordinating with 15 different partnering entities. Duran said that "in addition to the five partners that Vail Resorts and the National Forest Foundation are funding, the other partners provide us with either funding, in-kind labor, feedback on the project design, and lessons learned about the wildlife species we are concerned about. This project captures the importance of wildlife conservation and uses the best available science and stakeholder collaboration in the process."
The two species of sage grouse found in Colorado are both listed as "species of special concern" on the state's list of threatened and endangered species. The Greater Sage Grouse can be found in parts of Eagle County mainly for its winter habitat. In spring, these ground-nesting birds move north where there is suitable habitat in Routt County for their spring breeding, nesting, and summer habitat. In winter, the pungent green leafs of sagebrush account for 100 percent of the bird's diet, as well as providing shelter from winter weather and escape cover from predators like coyote, bobcat, badger, falcons, hawks and eagles. It's an intricate web and balance of resources that all depend on the well-being of the "sagebrush sea." These days, sustaining this balance requires active stewardship, and sometimes a chainsaw crew.
Getchen Van De Carr with the Rocky Mountain Youth Corps based out of Steamboat says "It may seem like a stretch to refer to chainsaw crews as "conservationists" yet these projects are evidence that training and skills with powerful machines can be used to preserve habitat.
The Greater Sage Grouse has been declining in population since pinyon and juniper trees have started to impede on the sage. Enter a chainsaw crew to restore the balance. Engaging young adults in habitat restoration instills a sense of ownership and value to the community. These personal growth outcomes are critical, especially today when unemployment and unhealthy lifestyles are impediments to so many young people's true development into tomorrow's leaders."
The Sagebrush Enhancement Project is one of three priority conservation partnership projects for the Eagle-Holy Cross Ranger District and the National Forest Foundation. The emphasis of these projects is threefold: to connect communities to the land, to enhance resilient ecosystems and healthy landscapes, and to create sustainable economies.
The National Forest Foundation is a nonprofit organization that works with the U.S. Forest Service and community-based organizations to restore and enhance National Forests. At least 62 million acres of the 193 million acre National Forest System are in need of restorative action due to damage from wildfire, insects, disease and natural disasters. As the nonprofit partner of the U.S. Forest Service, the National Forest Foundation engages people in programs that promote the health and public enjoyment of the National Forest System. The National Forest Foundation also administers private gifts of funds and land for the benefit of the National Forests.
Kim Langmaid Ph.D. lives in Vail and is the director of Colorado Programs for the National Forest Foundation. Contact her at email@example.com or visit http://www.nationalforests.org
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Remains discovered at Gypsum explosion site; Chatfield Corners residents allowed to return
Some residents of Gypsum's Chatfield Corners neighborhood were allowed to return home Friday afternoon following a Thursday explosion that destroyed a home in the subdivision.
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text:
Today's Trunkline 2012 presents to you tales of trouble for our President regarding Benghazi and his reelection chances as well as other tid-bits such as the potential names in Romney's future Administration, what Vice President Biden finds funny about breast cancer, na ew ad from the families of the victims of the Ft. Hood terrorist attack makes a plea to the President , a look at the dignity and grace of the Romneys, video of the hysterical speeches offered by Obama and Romney at the Al Smith dinner, and as always, much more
- Obama campaign is increasingly worried
- Benghazi is increasingly becoming the undoing of President Obama
- Fort Hood Victims Want President Obama to Finally Classify The Shooting Rampage A Terror Attack
- Romney circling around the soft underbelly of undefended Democratic states
- Is Obama Buying the Election with His Welfare Explosion?
- Dumb ass Joe Biden cracks jokes, laughs as woman in Florida tells breast cancer story (Video)
- So Much for Obama's New Era of Responsibility
- Names to Expect in a Romney Administration
- A Sign of Things To Come
- Romney's and Obama's remarks at the Al Smith dinner. If you haven't seen it yet, you must. It's hysterical!!
Filed under: Trunkline 2012 | Tagged: 2012 election news, al smith dinner video, Anthony Del Pellegrino, Benghazi is becoming the undoing of President Obama, biden laughs at breast cancer story, dick morris, election headquarters, election news, election news service, election news summary, election report, Fort Hood victims, Fort Hood Victims Want President Obama to Finally Classify The Shooting Rampage A Terror Attack, Intellectual Conservative, Is Obama Buying the Election with His Welfare spending, kempite, Lawrence Kudlow, Mitt Romney news, Names to Expect in a Romney Administration, national journal, national review, news summary, Obama campign is increasingly worried, Obama's New Era of Responsibility, online election news, political election news, political headquarters, political news, political report, potential members of the Romney administration. potential Romney cabinet officials, potential romney cabinet members, republican election news, Republican news, republican report, Republican Trunkline, Romney to go after solid Obama states, Romney's potential cabinet, romney's speech at the al smith dinner, the 2012 presidential election, The Dignity and Grace of the Romneys, The examiner, The Inquister, the race for the white house, trunkline 2012, White House 2012, who will romney pick for his cabinet, wordpress political blogs | Leave a comment »


text:
One of the greatest challenges advisors face is efficiently managing their clients' portfolios. Moreover, as the number of clients grow, unless we have a good system in place, the challenge may only get worse. Here's what I'm doing to stay on top of this all-important task.
Delegate or Do-It-Yourself?
As advisors, we are obligated to manage our clients' assets to the best of our abilities; some under a fiduciary capacity, some under a suitability requirement. We can do it ourselves or delegate this task to an outside firm. Giving this task to an outside firm would require that we have a great deal of trust in them, much as our clients had to have when they selected us as their advisor. At this point, I choose to manage client assets myself. However, I can imagine a day when I outsource this to a money management firm. If you are doing it yourself, a necessary prerequisite will be to divide your book into segments. Let's take a brief look at this.
Inch by Inch, It's a Cinch
One of the most important issues we can tackle is client segmentation. In other words, we need to divide our clients into groups, based on certain criteria, and determine the services we will provide to each group. Many of us, myself included, would like to provide the same services to all clients, but in reality, this is not feasible. Therefore, I have grouped my clients into A, B and C. Nothing startling there, I admit. What's important is that you create the groups and that the segmentation works for you.
I will do an internal account review monthly for clients in the A group. In short, I will review their performance and composition and send them a report. Here's the point. It's easier to eat an elephant one bite at a time. I have been trying to eat the whole thing all at once! By breaking it down into smaller pieces, it'll be easier to manage. I will also conduct an internal account review for clients in the B group on a quarterly basis and the C group annually. It's also important to put this on the calendar. Setting a reminder in your CMS will help you stay on top of this.
Client communication is an issue which is consistently at the top of client surveys. Maintaining regular communication with clients can also help foster trust and it tells them, "My advisor is looking out for me." And if you communicate based on your client segmentation, you're giving your "A" clients "A" service.
Take care and have a great week!


text:
Jeffry Ave, Jamaica, N.Y.
June 16, 1918
Dear brother Joe:
I suppose it will be some time before we get any mail from you but I know that you will be glad to hear from home, for when one is a distance from old New York they always like to know whether the old town is still on the map. I don't know whether you have heard of Fritz's latest scheme, but he is now operating a few submarines off our coast, but like the brave man he is, he is attacking a few small unarmed craft and is taking damn good care to keep away from anything that looks like a war vessel or a transport in convoy. Generally speaking he has made a fairly good mess of things. My boss, Paymaster Karker, left for duty overseas the other day and before he left he fixed things so that I can follow him later if everything else is O.K. I hope to be able to arrange matters, for I am really fond of him, as he is one of the most efficient officers in the Navy. My old team, the Yankees are going pretty good so far this season and I am pulling for them to win the pennant. Home Run Baker is pounding the cover off the ball and the rest of the team is following his example; the fans have nicknamed them "Murders Row" for the way the can slam that old ball.
Bill Zimmer, who formerly was quite a ball player here in town, I think you know him, was home from Camp Devens last Sunday. He shapes up pretty good as a soldier and says he likes the life and that he has gained 20 lbs. in weight. Doctor Hyland was around town in uniform yesterday. He has just been made a first lieutenant in the Medical Corps and leaves in a few days for some camp. Lieut. Fletcher, who lived down in Bergens house has also gone Over There.
I hope, Joe, that you are in good health and spirit, and that you are learning fast, so that when the proper time comes you will be able to fill any position of responsibility assigned you, for you are aware surely of our keen interest in your future.
I will close for now, dear brother, with the sincere wish that no matter what may happen you will always show the stuff you are made of and make us all feel proud of you.
With love and best wishes for all, and may God bless and protect you. I remain, as ever.
© Copyright 2011 by Lanny & Patti Brown, All Rights Reserved. No reproduction without permission.


text:
Brooklyn Watch Co. Bedford Brownstone Chronograph Grey Dial Mens Watch BW-308-E-104-BB
- Description
This Bedford Brownstone Chronograph Brooklyn Watch is operated by a Swiss Quartz movement that features date, day, hour, minute, and second. Black ion-plated stainless steel case with a black ion-plated stainless steel bracelet. Date display at the 4 o'clock position. Chronograph - sub-dials displaying: three - 60 second and 30 minute, day of the week. Grey dial with black hands and Arabic numeral and index hour markers.Brooklyn Features
- Scratch resistant Sapphitek crystal crystal.
- Delicately handcrafted B second hand
- B Emblem on the left side crown
Technical Data
Function: Chronograph, Date, Day, Hour, Minute, Second Movement: ISA 8172A Case Diameter: 44mm Case Material: Stainless Steel Crown: Pull / Push Crystal: Sapphitek Dial Color: Grey Bezel: Fixed Numerals: Arabic Numeral and Index Hands: Black Band Material: Black Ion-plated Stainless Steel Band Width: 20mm Buckle: Tang Water Resistance: 10 ATM - 100 Meters
- Return & Exchange
Brooklyn Watches is committed to making your shopping experience a success and offers free returns and exchanges on all web orders. If you are not 100% satisfied with your purchase, we will accept returns of unworn, unused merchandise for up to 30 days from purchase for a full refund or exchange. Proof of purchase, such as a copy of the original sales receipt or packing slip invoice, is required for reimbursement of the full purchase price. In all cases, refunds will be made in the form of the original payment.
To process a return or exchange and receive your Return Merchandise Authorization (RMA) number, please contact us via email at email@example.com
All returns and exchanges of Brooklyn Watch merchandise must be mailed to:
Brooklyn Watches Co.
Attn: Returns Dept.
140 58th Street
Unit 3B
Brooklyn, NY 11220
(Please note that this address is for mail-in returns / exchanges only.)
- When processing your return, we will refund the same credit card used to place the original order.
- When processing your exchange, we will first refund the same credit card a used to place the original order, then charge you for the merchandise you are wanting in the exchange. In the event the item you want in exchange for your original purchase is not available you will be notified via email.
- Shipping Policy
Brooklyn Watches offers two different shipping options: Standard and Next Day. Shipping is provided by UPS, and orders will be processed within two business days pending credit card approval.Standard:
Enjoy complimentary standard shipping on all orders. Arrives in 3 to 7 business days.
Standard delivery via UPS is available for orders with delivery addresses within the US and territories, excluding PO Boxes and Rural Routes. Orders being shipped by USPS, outside of the continental US or to APO/FPO address will arrive in 3-4 weeks, not the standard 3-7 days.
Next Day:
Next day shipping fee is $19.95.
Next day delivery is only available for orders with delivery addresses within the continental United States (excluding PO Boxes and Rural Routes).
Orders must be received by 12:00 pm CST in order to arrive on the next business day.
If a shipped order was lost or damaged, UPS or USPS must conduct an investigation. Please contact Brooklyn Watches to initiate a tracer within 10 business days of the item shipping from our warehouse. If the package is not located, UPS and USPS will process a claim for the package. Claims can take two weeks to process. When the carrier claims responsibility for the loss or damage of your shipped item(s), insurance reimbursement will be issued to Brooklyn Watches and the replacement item(s) will be shipped out. If the item(s) is no longer available, and a similar item is not wanted, Brooklyn Watches will gladly refund your original payment.
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Tying the knot is one of the most important moments in one's life. So it goes without saying that choosing the right gift is important, too! You can always play it safe and purchase a gift from the bridal registry that is sure to make the bride happy. Or, here are a few gift ideas that are sure to send the bride and groom into marital bliss.
Store Directory
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Dolfingers, 4600 Shelbyville Road, Suite #143, 502.895.3226
Southern Belle Boutique, www.thesouthernbelleboutique.net
Bittners, 731 East Main St., 502.584.6349


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In what became an emotional hearing on Capitol Hill, Gregory Hicks testified Wednesday that he and a defense attaché tried to send four more special forces to Benghazi and pleaded for air support -- but was told to stand down. NBC's Andrea Mitchell reports.
In a day of congressional testimony that once again found the Obama administration under fire, a trio of whistleblowers expressed frustration toward the government's response to the Sept. 11, 2012 assault against a U.S. diplomatic outpost in Benghazi, Libya, and its subsequent investigation into that incident.
The diplomatic officials appeared on Wednesday before the House Oversight and Government Reform Committee to describe a hasty and chaotic response to the attack, which left four Americans – including U.S. Ambassador Christopher Stevens – dead.
The witnesses said that the government was poorly prepared to weather the attack and was hesitant to respond, also contending that a subsequent review of the incident ordered by the State Department came up woefully short.
Sen. Lindsey Graham, R-S.C., joins Morning Joe to discuss Wednesday's House Oversight Committee hearing on the Sept. 11, 2012 Benghazi attacks that left four dead, including Amb. Chris Stevens.
The testimony included new details from Gregory Hicks, a career foreign service officer who served as the deputy chief of mission in Libya at the time of the attacks.
He painstakingly recounted frenetic efforts to communicate between besieged individuals in Benghazi, and the governments of Libya and the United States. And he relayed the frustration of special forces who were told to stand down in Tripoli – Hicks said he did not know who gave the order – from deploying to Benghazi.
"They were furious," Hicks told lawmakers on Capitol Hill. "I will quote Lt. Col. Gibson. He said, 'This is the first time in my career that a diplomat has more balls than somebody in the military.'"
IN DEPTH: Official: US Special Forces team wasn't allowed to fly to Benghazi during attack
Hicks joined two other witnesses in a hearing driven primarily by Republicans, who have zealously pursued the Benghazi incident based on suspicions that President Barack Obama and then-Secretary of State Hillary Clinton had been caught flat-footed by the attack, or worse, orchestrated a cover-up about the attack to benefit the president's re-election bid.
At no point did Hicks or his fellow witnesses – Mark Thompson, acting deputy assistant secretary for counterterrorism, and Eric Nordstrom, diplomatic security officer and former regional security officer in Libya – accuse the president or Clinton of having halted forces that might have assisted besieged diplomats in Benghazi. Democrats repeatedly pointed to testimony suggesting that reinforcements would have not have arrived in time, anyway.
But Republicans seized on several morsels of information, in particular Hicks's incredulity toward the administration's initial explanation, voiced by U.N. Ambassador Susan Rice, that the attack was the spontaneous outgrowth of protests related to an anti-Islamic video.
"I was stunned. My jaw dropped, and I was embarrassed," Hicks said of his reaction to Rice's appearances on a series of Sunday talk shows following the attack. He further testified that there were no indications of protests in Libya, and that at no time did they suspect that the Benghazi attack was related to protests.
Republicans also homed in on suggestions by Hicks that a top Clinton aide had reacted angrily when Hicks agreed to speak privately with GOP investigators looking into the Benghazi attack. Hicks said that Cheryl Mills, Clinton's chief of staff, called him "upset" about his conversation with the GOP lawmakers.
The witnesses also expressed their misgivings about the Accountability Review Board's (ARB) findings in a subsequent investigation into the government's response to the attacks. The ARB, the witnesses said, failed to interview senior enough leaders in the State Department.
The testimony prompted pointed responses from Ambassador Thomas Pickering, who co-authored the ARB, and allies of Clinton, the popular former secretary of state who's seen as a potential presidential candidate in 2016.
Sen. Bob Corker joins The Daily Rundown to discuss the latest with Syria, the investigation behind the attacks in Benghazi, and the rise of sexual assaults in the military.
"I believe the Accountability Review Board did its work well," Pickering, a coauthor of the report, said Wednesday afternoon on MSNBC. "I think the notion, quote, of 'a cover-up' has the elements of Pulitzer Prize fiction attached to it."
And Philippe Reines, a senior aide to Clinton, told NBC News that accusations that Mills interfered in an investigation into Benghazi "completely and utterly false."
Indeed, Democrats headed into the hearing warning against politicization of the Benghazi incident.
"I am not questioning the motives of our witnesses," said Maryland Rep. Elijah Cummings, the top Democrat on the committee, at the outset of the hearings. "I am questioning the motives of those who want to use their statements for political purposes."
His admonition didn't stop many Republicans from plowing ahead with their questions.
"It's one of great mysteries," said Rep. Jason Chaffetz, R-Utah, of questions as to why terror response forces were not ordered into action on Sept. 11. "Here we have this expertise, we've invested heavily in it, they tabletop it, they understand it, this is exactly what they train for and they were never asked to go into action."
But while many Republicans appeared eager to keep Benghazi alive as a political issue, not all Republicans seemed as concerned about the issue, or the Obama administration's forthcoming.
"I've been able to read all the cables, I've seen all the films. I feel like I know what happened in Benghazi; I'm fairly satisfied," said Sen. Bob Corker, Tenn., the top Republican on the Senate Foreign Relations Committee, on MSNBC. "I'm fairly satisfied."
This story was originally published on Wed May 8, 2013 11:02 AM EDT


text: FREE: Visual Studio Express 2012 for Windows Desktop September 13, '12 Comments [32] Posted in Open Source | VS2012 Sponsored By When the Visual Studio 2012 free versions were originally announced the first thing I noticed was that they had switched from a language-specific model (C# Express, VB Express, etc) to a target-specific model (Windows 8, Web). However, I was very surprised (and disappointed) that there was no free way to make Console Apps, or Windows Forms apps, or anything for the Windows Desktop. I wasn't the only one who thought this was a problem. Shortly thereafter (with a lot of people pushing) Soma announced there would be a "Windows Desktop" Express version for free. He said: ...we heard from our community that developers want to have for Windows desktop development the same great experience and access to the latest Visual Studio 2012 features at the Express level. Today, I'm happy to announce that we will add Visual Studio Express 2012 for Windows Desktop to the Visual Studio 2012 family. This will bring to the Visual Studio Express family significant new capabilities that we've made available in Visual Studio 2012 for building great desktop applications. I'm glad folks realized that no-cost desktop software development is important. Open Source projects need free tools like the Express SKUs. Even better that the the Express Desktop SKU gets the new 2012 features as well. Today Visual Studio has made Visual Studio Express 2012 for Windows Desktop available and you can go download it now free. The best part is that this one SKU supports C++, C#, and Visual Basic together. With this one free version you can make WinForms, WPF, Console or Class Libraries with any or all of Visual Basic, C#, as well as Win32 projects, class libraries, and CLR apps using C++. You can also, of course, combine projects over multiple languages into a single solution. You can target both .NET 4.0 and 4.5. NOTE: You might wonder, what about a free F#? Why isn't F# included? We've got a free download to add F# support to the free Visual Studio Express 2012 for Web! Related Links Download page for Visual Studio 2012 for Windows Desktop (Web Installer or ISO) Product page and Details for VS2012 for Windows Desktop F# Tools for Visual Studio 2012 Express for Web (you *don't* need this if you have Pro already, this is just for Express for Web) While Express SKUs don't allow arbitrary add-ins (you need Pro for that) the free SKU does include Unit Testing, Code Analysis, as well as the NuGet package manager. It's a bit of a nice coup for my little group that NuGet is now included in ALL Visual Studio 2012 SKUs, even Express ones. Package management is finally happening in .NET. In the screenshot below I've added a C++ Console app, a Window Forms C# app and a C# Console to a single solution in VS2012 using Express for Windows Desktop. Just to illustrate one of the reasons I think a free "Desktop" SKU is so important, I wanted to share a cool open source project I found recently called Topshelf. It's a library for making Windows Services easier to write using .NET. It's up on GitHub under an Apache license. The easiest way to get Topshelf is with NuGet with simply "install-package Topshelf". Topshelf has some impressive documentation as well, especially for an open source project! Here's a simple Windows Service with a basic heartbeat timer using Topshelf: public class TownCrier{ readonly Timer _timer; public TownCrier() { _timer = new Timer(1000) {AutoReset = true}; _timer.Elapsed += (sender, eventArgs) => Console.WriteLine("It is {0} an all is well", DateTime.Now); } public void Start() { _timer.Start(); } public void Stop() { _timer.Stop(); }}public class Program{ public static void Main() { HostFactory.Run(x => //1 { x.Service<TownCrier>(s => //2 { s.ConstructUsing(name=> new TownCrier()); //3 s.WhenStarted(tc => tc.Start()); //4 s.WhenStopped(tc => tc.Stop()); //5 }); x.RunAsLocalSystem(); //6 x.SetDescription("Sample Topshelf Host"); //7 x.SetDisplayName("Stuff"); //8 x.SetServiceName("stuff"); //9 }); //10 }} Topshelf even has nice Log4net and NLog integration. Anyway, this is just the kind of powerful, useful, and interesting open source library that could be helped by a free Express SKU for Desktop. I'm not involved directly (yet ;) ) in making decisions this high up, but I (and many, many others) inside and out continue to advocate for balance. In this case I'm very glad that the decision was made to ship this SKU and I hope you all find it useful whether you work in open source or in education. There's more details over at the Visual Studio blog and the team is watching the comments. This week's sponsor: Be part of GENERATION APP. Your Idea. Your App. 30 Days. Begin your 30-day journey to create a Windows Store style app and talk 1-on-1 with a Windows 8 app development pro. Get started today. « ASP.NET Web Forms DynamicData FieldTempl... | Blog Home | Dick and Jane: Jedi Masters » About Scott Scott Hanselman is a former professor, former Chief Architect in finance, now speaker, consultant, father, diabetic, and Microsoft employee. He is a failed stand-up comic, a cornrower, and a book author. About Newsletter Sponsored By Hosting By Comments [32] Share on: Twitter, Facebook, Google+ or use the Permalink


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The kids are running for the ferry to Lilycove and jump on the boat they see leaving, only to discover it's actually a boat to Bomba Island. Once they arrive on the island, they meet a young Trainer named Jimmy and his Wartortle, Charmeleon and Ivysaur. Jimmy is a student at the Pokemon Battle Judge Trainin Institute, located on the island.
Professor Serena from the institute gets Ash and Brock to help out in her class by staging a Double Battle which Jimmy will judge. Ash uses Swellow and Corphish, while Brock uses Mudkip and Lombre. The battle is fierce, and after Mudkip and Lombre both use Water Gun on Corphish simultaneously, Jimmy mistakenly declares Corphish unable to battle.
That night, Ash and friends find Jimmy outside talking to his Pokemon. Jimmy explains how he met all three of them by accident, and even got a Cascade Badge--also by accident. Team Rocket interrupts and takes all three of the Pokemon, causing Jimmy to run after them. Seeing his determination, Wartortle evolves into Blastoise and uses Hydro Pump to free the others. Team Rocket is the defeated as usual.
Serena tells Jimmy that he has to leave school now, but not because he's a bad student, but because he'd make a better Trainer. Jimmy heads back to Lilycove to start his Pokemon journey over, while Ash and friends continue on to Mossdeep City.
Dub edits
- The sandwich that rolls down the hill into Ivysaur's mouth in the flashback was an onigiri in the original.
|This article has an incomplete plot/synopsis.
Reason: N/A
Please help the Pokémon Wiki by expanding it.
|This article is an anime stub.
Please help the Pokémon Wiki by expanding it.


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Gone are the days when automakers wouldn't use anything not designed in their own studios (about forty years gone, if we're being honest). The powertrain of Dodge's new 2007 Caliber, for example, is a figment of globalization worthy of champagne and tongue-kisses from George Soros. For one, the Caliber's CVT2 continuously variable transmission (pictured), a DiVinciesque system of belts, pulleys and cogs, was created by Nissan's Jatco, Ltd. (CVT systems, as you may know, allow a car's engine to stay in its optimum RPM range, notwithstanding its velocity.)
One notable feature of the CVT2 application, is software that mimics the feel of a traditional transmission, squelching the instant rise in RPMs that could give first-time CVT operators the sensation of slipping on banana peels.
What's more, the Caliber's all-wheel-drive system — with variable torque output for front and rear — was developed with Mitsubishi. (It's Chrysler Group s first electronically controlled AWD system.) Plus, VW will supply a 2.0-liter direct-injection turbodiesel engine that will be available in overseas markets.
More on Jeep's Dodge Caliber Platform Crossovers; Jeep Releases Images of Frankfurt Concepts [internal]


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4 Tips for Caregivers During the Chaos of the Diagnosis Have a loved one who is newly diagnosed? Cameron Von St. James, husband and caregiver, to Heather, a mesothelioma survivor, discusses tips to deal with the chaos of a diagnosis.
Caregiver Stress & Burnout: Tips for Regaining Your Energy, Optimism, and Hope
In partnership with Harvard University, HelpGuide.org offers pragmatic advice for maintaining your health and wellbeing while taking care of your loved one.
The Challenge of Caregiving + 5 Ways to Approach It As You Would Yoga For those who enjoy yoga and its philosophy, this article is "enlightening".
Inner Strength Meditation for Caregivers - YouTubeThose who wish to try guided meditation only have to go as far as their tablet or computer. This is just a sample of what is available searching "YouTube Guided Meditation for Caregivers".
How to Relieve the Stress of Caring for an Aging Parent
This TEDX talk is one of dozens on the subject of caregiving and the "sandwich" generation. Click on it and many more will appear for viewing.
Caregiver stress fact sheet | womenshealth.gov The US Department of Health and Human Services, Office of Women's Health has compiled worksheets, tips and resources for caregivers from public and private-sector sources. Through this site you can also navigate to Medicare and Medicaid pages with information about disability and other reimbursement programs.
Eldercare Locator
This site allows the user to search by zip code for eldercare agencies, both public-sector and nonprofit. It is a service of the U.S. Administration on Aging.
Alzheimer's Association - Community Resource Finder for Caregivers
Alzheimer's disease can effect individuals as young as 30-something and those who develop it may live for decades with progressively worsening symptoms. Caregivers can find help taking care of loved ones living with early, middle and advanced-stage Alzheimer's as well as support groups in their neighborhood.
Mesothelioma Caregiver Support | mesothelioma.comMesothelioma is a rare and aggressive cancer, that can affect anybody those who have been exposed to asbestos in their lifetime. This disease has a long latency period of 10-50 years. Those who are diagnosed are typically given 12-21 months to live. Caregivers of mesothelioma patients play a crucial role, and often times are there to support them in their end of life.
Caregiver Action Network
One of the most robust sources of caregiver Information online comes from the Caregiver Action Network (CAN). A national, non-profit advocacy organization, CAN offers a variety of links to specific areas of interest and a good search function.
Western Reserve Area Agency on Aging (WRAAA)WRAAA is a state designated agency that serves older adults and disabled residents in Cuyahoga, Geauga, Lake, Lorain and Medina counties. Search this site for access to community services, public events and federal, state and local programs and benefits information.
Hospice of the Western Reserve is a community-based 501(c)(3) non-profit hospice, tax ID: 34-1256377 Your donation is tax-deductible as permitted by law.
Hospice of the Western Reserve provides palliative and end-of-life care,
caregiver support, and bereavement services throughout Northern Ohio. In
celebration of the individual worth of each life, we strive to relieve
suffering, enhance comfort, promote quality of life, foster choice in
end-of-life care, and support effective grieving.
17876 St. Clair Avenue
Cleveland, Ohio 44110
Hospice of the Western Reserve provides palliative and end-of-life care,
caregiver support, and bereavement services throughout Northern Ohio. In
celebration of the individual worth of each life, we strive to relieve
suffering, enhance comfort, promote quality of life, foster choice in
end-of-life care, and support effective grieving.
Hospice of the Western Reserve All Rights Reserved.


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Rick Gorka, mind your manners. Gorka, Mitt Romney's press spokesperson, is in the news for losing his temper on Romney's already rather problematic foreign tour. Having generated negative headlines in the UK and in the Middle East, the Romney campaign now finds itself in the media spotlight again because Gorka, its spokesperson, told journalists to "kiss [his] a**" and "shove it." Now, is that any way to behave–especially for a spokesperson?
On the other hand, who can blame him? This tour has not been the most wildly successful of trips. Gorka's not-so-friendly advice to the press corps came after journalists fired questions from behind a rope. Reporters from the New York Times, CNN, and Politico.com yelled questions about Romney's European gaffes, wanting Mitt to respond. Gorka, on his last nerve, told them exactly how he felt.
The media went wild, Mitt looked embarrassed, and Gorka made personal apologies to the journalists concerned the next day. Any harm done? Hard to say. Everyone has an off day, and if Gorka has media relationships worth anything, one might hope this will be forgiven. What seems the bigger issue is the media's complaint about access to Mitt himself, and that might be worth a rethink.
The PR Verdict: "C" (Distinctly OK) for Rick Gorka. At least he said "sorry" quickly and turned the page, but is there a wider issue at play?
The PR Takeaway: Apologizing is the easy part. The persistent complaint from the media is that Romney has simply been unavailable for much of his grand tour. In this latest blowup, the journalist yelled at Gorka, "We haven't had another chance to ask a question!" Since Romney's tax issues put his PR team on alert, the media have been complaining about restricted access. Romney did not address members of the press flying with him on any of the three charter flights–two that lasted more than four hours. One sure way to annoy the media? Ignore them. And for that, Gorka may be apologizing for some time to come.